System and method for flat panel detector gel and blot imaging

ABSTRACT

A system and method for generating a digital image in fluorescence gel imaging is disclosed. The method includes providing a gel sample and placing the gel sample on a flat panel detector having array of photodiodes and transistors that collect light generated from the gel sample. The gel sample is illuminated using a light source integrated into the flat panel imaging system and light emitted by the gel sample responsive to an excitation of the gel sample by light provided by the light source is then collected, with the light emitted by the gel sample being collected by the array of photodiodes of the flat panel detector and converted to electric charges to generate light data. The light data is then processed to generate a digital image of the gel sample.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 14/245,041, filed Apr. 4, 2014, thedisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Embodiments of the invention relate generally to gel and blot imagingand, more particularly, to a system and method for performing gel andblot imaging using a flat panel imaging system.

Gel electrophoresis and electroblotting are commonly used techniques forthe separation and analysis of macromolecules (DNA, RNA and proteins)and the subsequent transfer of such macromolecules onto a membrane,respectively, that then enables further analysis of these macromoleculesusing probes, such as specific antibodies, ligands, or stains, that canand/or drive a reaction and produce a color blot (e.g., Western blotimaging and Southern blot imaging). Several detection techniques may beemployed in gel and blot imaging for DNA and/or protein analysis,including the detection, recognition, and quantification of specificmacromolecules in a sample of tissue homogenate or extract. Suchtechniques include fluorescent detection, chemiluminescent detection,and colorimetric detection. In fluorescent detection, a fluorescentlylabeled stain or probe is excited by light and the emission of theexcitation is then detected by a photosensor (e.g., a charge coupleddevice (CCD) camera) that captures a digital image of the gel/blot andallows further data analysis, such as molecular weight analysis and aquantitative western blot analysis. In chemiluminescent detection, ablot is incubated with a substrate that will luminesce when exposed to areporter on the antibody—with the light that is generated being detectedby photographic film to create an image of the blot thereon or by CCDcameras to capture a digital image of the blot.

The performing of fluorescent detection, chemiluminescent detection,and/or colorimetric detection according to existingtechniques—specifically with respect to the use of film emulsion and/orCCD cameras to capture images—presents some drawbacks and limitations.For example, film emulsion is the conventional detection medium forchemiluminescent detection, but is characterized by non-linear responseand limited dynamic range requiring multiple exposures, therebyresulting in a time-consuming and expensive imaging procedure. Asanother example, as chemiluminescent signals generated from the blotsare normally weak and time-varying, relatively fast exposure (e.g., onthe order of a minute), low noise, and high light detection efficiencyis required for accurate image capture when using CCDs. Thus,limitations of the CCDs regarding operation at a low frame rate (due tothe inherent sequential read-out thereof) and low temperature (toachieve a reasonable noise level) present challenges in accuratelycapturing the chemiluminescent signals. Still further, CCDs require ahigh efficiency optical lens to focus the large blot to small CCD chips(˜1 cm²)—with the optical lens adding to the cost of the high-end CCDs,increasing the size and vertical space of the imaging device (due to thelarge working distance of the CCD camera), and also causing problemswith regard to light collection efficiency (due to the large workingdistance). Yet still another drawback of image capture via CCD is thatthe capturing of images can take approximately 3-20 minutes—depending onthe desired exposure.

Other more recent attempts to provide a system that captures a digitalimage of the blot include a C-digit system released by LICOR Biosciencesthat utilizes a linear scanner with sixteen linear sensors. The linearscanner combines short working distance (like film emulsion) to maximizelight collection efficiency and multiple small low cost linear sensorarrays to meet the data acquisition time requirement, but the scan timeto scan the large area is still around multiple minutes per pass (i.e.,6-12 minutes). Additionally, there is a concern that during the scanningtime (on order of 10 minutes), the transient behavior of thechemiluminescence in the blot itself will be changing. As such—as thelinear scan is happening—the intensity at the beginning of the scan willbe higher than the intensity of at the end of the scan (bottom of thescan), therefore introducing an artificial gradient in the measurement.

Therefore, it would be desirable to provide a system and method forimage acquisition in gel and blot imaging that overcomes theaforementioned drawbacks of conventional imaging techniques andassociated systems. It would also be desirable for such systems andmethods to provide improved performance in regards to sensitivity,dynamic range, exposure time, and quantum efficiency, while eliminatingcostly high-efficiency imaging optics such as are used with existing CCDimage sensors, so as to provide a system at a reduced cost and size.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, a method for generatinga digital image in fluorescence detection gel imaging includes providinga gel sample having a gel and a sample of macromolecules therein andplacing the gel sample on a flat panel detector of a flat panel imagingsystem, the flat panel detector comprising an array of photodiodes andtransistors that collect light generated from the gel sample. The methodalso includes illuminating the gel sample using a light sourceintegrated into the flat panel imaging system and collecting lightemitted by the gel sample responsive to an excitation of the gel sampleby light provided by the light source, with the light emitted by the gelsample being collected by the array of photodiodes of the flat paneldetector and converted to electric charges to generate light data. Themethod further includes processing the light data to generate a digitalimage of the gel sample, the processing and image generation beingperformed by an image reconstructor separate from or incorporated intothe flat panel imaging system.

In accordance with another aspect of the invention, a method ofgenerating a digital image in fluorescence detection gel imagingincludes providing a gel sample labeled with a fluorescent reagent, thegel sample having macromolecules labeled by the fluorescent reagent. Themethod also includes positioning the gel sample within a flat panelimaging system to provide for capturing of a digital image of the gelsample, the positioning of the gel sample within the flat panel imagingsystem further including placing the gel sample on a flat panelmatrix-based light sensor comprising an array of photodiodes andtransistors and closing a lid of the flat panel imaging system to createa closed environment for capturing of the digital image. The methodfurther includes illuminating the gel sample using a light sourceintegrated into the lid of the flat panel imaging system so as to excitethe fluorescent reagent causing the gel sample to generate fluorescentlight and detecting the fluorescent light emitted by the gel sampleusing the flat panel matrix-based light sensor, with the fluorescentlight being collected by the array of photodiodes, converted to electriccharges, and subsequently converted to digital signals. The method stillfurther includes providing the digital signals to an image reconstructorto process the digital signals and generate a digital image of the gelsample.

Various other features and advantages will be made apparent from thefollowing detailed description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate preferred embodiments presently contemplated forcarrying out the invention.

In the drawings:

FIG. 1 is an elevated perspective view of a flat panel imaging system,including a flat panel detector, according to an embodiment of theinvention.

FIG. 2 is an exploded sectional view of the flat panel detector of FIG.1 taken along line II-II, according to an embodiment of the invention.

FIG. 3 is an elevated prospective view of the flat panel detector ofFIG. 1 removed from a protective base portion, according to anembodiment of the invention.

FIG. 4 is a schematic view of an amorphous silicon photodetector arrayof the flat panel detector of FIG. 1, according to an embodiment of theinvention.

FIG. 5 is a flowchart illustrating a technique for digital imageacquisition of a chemiluminescence blot using the flat panel imagingsystem of FIG. 1, according to an embodiment of the invention.

FIG. 6 is an elevated perspective view of a flat panel imaging system,including a flat panel detector, according to an embodiment of theinvention.

FIG. 7 is an exploded sectional view of the flat panel detector of FIG.6 taken along line VII-VII, according to an embodiment of the invention.

FIG. 8 is a flowchart illustrating a technique for digital imageacquisition of a fluorescence blot using the flat panel imaging systemof FIG. 6, according to an embodiment of the invention.

FIG. 9 is an elevated perspective view of a flat panel imaging system,including a flat panel detector, according to an embodiment of theinvention.

FIG. 10 is an exploded sectional view of the flat panel detector of FIG.6 taken along line X-X, according to an embodiment of the invention

FIG. 11 is a flowchart illustrating a technique for digital imageacquisition of a colorimetric blot using the flat panel imaging systemof FIG. 9, according to an embodiment of the invention.

FIG. 12 illustrates images acquired of a cell lysate sample using eachof a flat panel imaging system, CCD-based imaging system, and a C-Digitimaging system.

DETAILED DESCRIPTION

Embodiments of the invention relate generally to gel and blot imagingand, more particularly, to a system and method for performing gel andblot imaging using a flat panel imaging system. The flat panel imagingsystem is a two-dimensional light sensitive image detector array whichprovides a digital image of the light collected on the detector surface.According to embodiments of the invention, the flat panel imaging systemmay be specifically constructed to function under chemiluminescence,absorbance (colorimetric), and fluorescence gel and blot imaging modes.

Referring to FIGS. 1-4, a flat panel imaging system 10 for use in a geland blot digital image acquisition is provided according to anembodiment of the invention. FIG. 1 provides an elevated perspectiveview of the flat panel imaging system 10 generally including an outerhousing 12 that houses a flat panel detector 14 therein to surround andprotect the physical light receptors, electronic detection equipment andassociated electronics of the flat panel detector 14. The outer housing12 includes a base portion 16 that encases the flat panel detector 14and a lid 18 that, according to one embodiment, is hinged to the baseportion 16 so as to be selectively opened and closed with respect to thebase portion to provide a “closed environment” to exclude externalsources of light for performing of a gel/blot image acquisition.

The flat panel detector 14 of the flat panel imaging system 10 functionsas the light detection device in the gel or blot image acquisition. Inperforming the image acquisition, a “gel sample” or “blot sample” 20 isplaced directly onto the imaging surface of the flat panel detector 14,such that photons generated during the image acquisition are directlyand efficiently collected from the gel/blot sample. The “gel sample” isunderstood to refer to an agarose/polyacrylamide gel alone (withprotein/DNA/RNA sample therein), while the “blot sample” is understoodto refer to an macromolecules (i.e., protein/DNA/RNA sample therein)transferred from the gel onto a membrane. The flat panel detector 14provides a digital image of the light collected on the detector surface,with the digital image being a quasi-stationary image with desirablesignal-to-noise ratio.

Referring now to FIG. 2, an exploded sectional view of the flat paneldetector 14 taken along line II-II of FIG. 1 is provided to betterillustrate a construction of the flat panel detector. As shown, the flatpanel detector 14 includes a top protective layer 22 that providesprotection to the components of the detector and that also receives thegel or blot directly thereon. Photons generated during the gel/blotimage acquisition pass through protective layer 22 and are absorbed byan array of photodetectors (i.e., photodiode/transistor array) that,according to an exemplary embodiment, is formed from amorphous siliconpanel 24. While the array of photodetectors is described hereafter asbeing formed of an amorphous silicon 24, it is recognized thatpoly-silicon, an organic photodiode, or crystal silicon technology couldinstead be employed. As an example, in an embodiment where an organicphotodiode is employed, the organic photodiode material may include anelectron blocking layer including aromatic tertiary amines and polymericaromatic tertiary amines, a mixture of a donor material containing a lowbandgap polymer, and an acceptor material containing a fullerenematerial.

The photodiode/transistor array of the amorphous silicon panel 24receives and converts photons into a plurality of representative imagedata values 26. Image data values 26 are received in analog form byinterconnect electronics 28 and output therefrom as analog image data30. Amorphous silicon panel 24 and interconnect electronics 28 areformed on silicon glass substrate 32 through semiconductor technologyknown in the art. For example, in fabrication, eleven layers ofamorphous silicon, various metals, and insulators are deposited byplasma enhanced chemical vapor deposition (“PECVD”), sputtering andmeniscus coating to form field effect transistors (“FETs”), diodes,interconnects, and contacts. Together, the protective layer 22,amorphous silicon panel 24, interconnect electronics 28, and glasssubstrate 32 form a flat panel detector 14.

With respect to the top protective layer 22, the layer is constructed tospecifically accommodate placement of a gel or blot 20 thereon andprovide for accurate photon capture of the gel/blot. The protectivelayer 22 is thus formed so as to be transparent, sufficiently hard so asto resist scratching, and chemically resistant so as to allow wipe-downthereof with cleaning solvents after removal of a gel/blot uponcompletion of a digital image acquisition. According to embodiments ofthe invention, the protective layer 22 may be constructed of glass,mylar, or another suitable thin, tough plastic, or may be a combinationof both glass and plastic, where the plastic top sheet is a replaceablelayer. It is also recognized that, rather than a replaceable layer beingincluded on protective layer 22, a removable protective layer orprotective sleeve could be utilized to protect/enclose the blot sample(i.e., the membrane on which the sample is provided). In eachembodiment, the surfaces of protective layer 22 can also be coated witha conductive polymer (PDOT) or indium tin oxide (ITO), for example, soas to prevent artifacts or damage that might occur if statically chargedsamples (e.g., saran wrap) are placed on the detector.

According to an exemplary embodiment, the protective layer 22 isconstructed as a “thin” layer having a thickness of ˜25-75 um (e.g., 50um) in order to prevent light spreading and maintain good spatialresolution, with the protective layer 22 providing for optimaltransmission of photons therethrough so as not to degrade the modulationtransfer function (MTF) of the amorphous silicon panel 24. Theprotective layer 22 also provides thermal isolation between the gel/blot20 and the amorphous silicon panel 24, which is desirable as it isrecognized that the placement of cold liquid gels/blots in contact withthe imager—coupled with the long acquisition times (on the order ofminutes) that might be present in, for example, western blot imaging—cancause local changes in temperature, which in turn affect the leakagecurrent in the photodiodes, which can cause image artifacts.Additionally, the protective layer 22 may incorporate an anglediscriminating film thereon to increase contrast and decrease crosstalkof light received by the flat panel detector 14.

As can be seen in FIG. 2, the flat panel detector 14 does not include ascintillator material therein that is often found in such detectors—suchas in flat panel detectors used for various x-ray imaging applications.As the detector 14 acquires photons/light emissions directly from a gelor blot sample via use of the photodiodes, no scintillator is requiredfor converting x-ray/radiation into photons prior to receiving of thephotons by the photodiodes. Accordingly, the flat panel detector 14 isspecifically constructed for use in capturing digital images ofmacromolecules (e.g., proteins, DNA, RNA) that are analyzing via gel andblot imaging, microtiter plate imaging, etc.

Referring now to FIG. 3, an elevated prospective view of the flat paneldetector 14 removed from base portion 16 (FIG. 1) is provided. Asillustrated in FIG. 3, the top protective layer 22 covers the amorphoussilicon panel 24, with the amorphous silicon panel being comprised of anarray of photo cells or pixels 34 that convert light photons received onthe detector surface during gel and blot imaging to electrical signalsthat are representative of the number of photons or the intensity ofradiation impacting individual pixel regions of the detector surface.Row electrodes 36 and column electrodes 38 are connected to the pixels34—with each pixel being generally defined at a row and column crossing,at which a row electrode or scan line 36 crosses a column electrode ordata line 38. Contact fingers 40 are formed for receiving signals fromthe column electrodes 38, and contact leads 42 are provided forcommunicating the signals between the contact fingers 40 and readoutelectronics (not shown) that convert analog signals generated by thepixels 34 to digital values that can be processed, stored, and displayedfollowing reconstruction of an image.

As best illustrated in FIG. 4, the array elements or pixel regions 34are organized in rows and columns 36, 38, with each pixel 34 including aphotodiode 44 and associated thin film transistor 46 (TFT). The cathodeof each diode 44 is connected to the source of the transistor 46, andthe anodes of all diodes 44 are connected to a negative bias voltage 48.The gates of the transistors 46 in each row are connected together andthe row electrodes 36 are connected to scanning electronics 50 (i.e.,row drivers) described in further detail below. The drains of thetransistors 46 in a column are connected together and an electrode ofeach column 38 is connected to readout electronics 52. In operation, thephotodiodes 44 are biased by way of the negative bias voltage 48 anddischarged at the appropriate time by way of transistors 46, with thetransistors 46 controlling electrical discharge from the appropriatecorresponding columns 38. The rows 36 and columns 38 of pixels 34 definean image matrix 54, having a height and width of desired size. For theflat panel detector 14, which is utilized for gel and blot imaging, theimage matrix may be constructed to have dimensions of approximately40×40 cm, with an array of 2048 columns×2048 rows at 200 μm pitch,according to one embodiment. It is recognized, however, that the flatpanel detector 14 may be constructed to have different dimensions and adifferent array size at a different pitch, such as pixels at a 100 μmpitch or 50 μm pitch, for example. In general, the size of the panel isable to accommodate up to four mini gels/membranes or one largegel/membrane, such that flexibility for particular imaging needs orrequirements can be easily met.

Each of the rows and columns of pixels 36, 38 is coupled to a row bus 56and column bus 58, respectively. The row bus 56 includes a plurality ofconductors for enabling readout from various columns of the detector, aswell as for disabling rows and applying a charge compensation voltage toselected rows, where desired. The column bus 58 includes additionalconductors for reading out the columns while the rows are sequentiallyenabled. The row bus 56 is coupled to a series of row drivers 50, eachof which commands enabling of corresponding row 36. Similarly, readoutcircuitry or electronics 52 is coupled to column bus 58 for reading outall columns 38. According to one embodiment, in response to sequentialtrigger signals from row drivers 50, all columns are simultaneously readout by readout electronics 52.

As mentioned above, a thin film transistor 46 is provided at eachcrossing location for each photodiode of each pixel region 34. As eachrow 36 is enabled by row drivers 50, signals from each photodiode 44 maybe accessed via readout circuitry 52, and converted to digital signalsfor subsequent processing and image reconstruction—such as by way of animage reconstructor 60 provided separately from the flat panel imagingsystem 10. While image reconstructor 60 is shown separate from flatpanel imaging system 10 in FIG. 4, it is recognized that in anotherembodiment the image reconstructor 60 could be incorporated into theflat panel imaging system 10.

According to embodiments of the invention, the flat panel imaging system10 generally described in FIGS. 1-4 may be utilized for imaging geland/or blot samples of macromolecules (DNA, RNA and proteins), withimaging being performed according to one of various detectiontechniques, including chemiluminescence detection, fluorescentdetection, and colorimetric detection. Analysis of standalone gelsamples and blot samples (e.g., western blot imaging and southern blotimaging, for example) may be performed using the flat panel imagingsystem 10.

According to one embodiment, the flat panel imaging system 10 isutilized for chemiluminescence Western blot imaging. Accordingly, asshown in FIG. 1, the lid 18 of the flat panel imaging system 10 isconstructed as a “dark lid” that provides for an efficient capture oflight emitted from the chemiluminescent reaction in the blot 20 by theflat panel detector 14. The dark lid 18 mates with the base portion 16such that the outer housing 12 forms a light-tight box within which adigital image acquisition of the chemiluminescence blot imaging can beperformed.

Referring now to FIG. 5, and with continued reference back to FIGS. 1-4,a technique 62 of image generation for chemiluminescence blot imaging isillustrated according to an embodiment of the invention—with thetechnique being described for a Western blot technique. The technique 62begins at STEP 64 with the providing of a blot sample 20 that isprepared in accordance with a manner commonly known in the art. Inproviding the blot sample 20, proteins of the sample are first separatedusing gel electrophoresis—with the separation of proteins being byisoelectric point (pI), molecular weight, electric charge, or acombination of these factors, for example. The gel electrophoresis mayemploy polyacrylamide gels and buffers loaded with sodium dodecylsulfate (SDS), for example, to maintain polypeptides in a denaturedstate once they have been treated with strong reducing agents to removesecondary and tertiary structure (e.g., disulfide bonds [S—S] tosulfhydryl groups [SH and SH]) and to allow separation of proteins bytheir molecular weight. Sampled proteins become covered in thenegatively charged SDS and, upon applying of a voltage along the gel,move to the positively charged electrode through the acrylamide mesh ofthe gel. The proteins travel only in one dimension along the gel formost blots, with proteins migrating through the gel at different speeds(rates of advancement) dependent on their size—with the different ratesof advancement (different electrophoretic mobilities) separating theproteins into bands.

In order to make the proteins accessible to antibody detection they aremoved from within the gel onto a membrane made of nitrocellulose orpolyvinylidene difluoride (PVDF), for example, such as by way ofelectroblotting—with the proteins maintaining the organization they hadwithin the gel. In performing the antibody detection process, themembrane is “probed” for the protein of interest with a modifiedantibody which is linked to a reporter enzyme that—when exposed to anappropriate substrate—drives a colorimetric reaction and produces acolor. The antibody detection is typically a two-step process. In afirst step, a dilute solution of primary antibody (generally between 0.5and 5 micrograms/mL) is incubated with the membrane under gentleagitation. Typically, the solution is composed of buffered salinesolution with a small percentage of detergent, and sometimes withpowdered milk or BSA. After rinsing the membrane to remove unboundprimary antibody, the membrane is exposed to another antibody (i.e., asecondary antibody) directed at a species-specific portion of theprimary antibody. The secondary antibody is usually linked to biotin orto a reporter enzyme such as alkaline phosphatase or horseradishperoxidase (HRP), which means that several secondary antibodies willbind to one primary antibody and enhance the signal.

In order to generate chemiluminescence in the blot sample 20, asubstrate molecule is then provided with which the enzyme in thesecondary antibody reacts—i.e., the substrate molecule will be convertedby the enzyme to a colored reaction product or luminescence that will bevisible on the membrane, with the colored reaction product orluminescence being produced in proportion to the amount of protein. Asan example, in an embodiment where a HRP is the enzyme in the secondaryantibody, a luminol-based substrate is applied to produce achemiluminescent signal released in the form of light. In the presenceof HRP and a peroxide buffer, the luminol oxidizes and forms an excitedstate product that emits light as it decays to the ground state. A blotsample 20 that generates a chemiluminescent signal released in the formof light is thus provided at STEP 64.

Referring still to FIG. 5, in a next step of technique 62, the blotsample 20 is placed onto the flat panel detector 14 of the imager 10, asindicated at STEP 66. More specifically, the sample 20 is placed on thetop protective layer 22 of the flat panel detector 14 such that it is indirect contact therewith. Accordingly, the blot sample 20 is positionedimmediately adjacent to the amorphous silicon photodetector array 24 ofthe flat panel detector 14. Upon placement of the blot sample 20 on theflat panel detector 14, the dark lid 18 of the flat panel imaging system10 is closed at STEP 68 and acquisition/capture of the chemiluminescentsignal (i.e., light) emitted from the blot sample 20 via the flat paneldetector 14 is commenced at STEP 70. Light emitted from the blot sample20 is converted to electric charge and stored in the photodiodes 44 ofthe photodetector pixels 34. The charge is read out by activating thethin film transistors 46 associated with the photodiodes 44, with theread-out time being adjustable as desired for properly detecting thechemiluminescent light signal emitted from the blot sample 20.

The read-out of the charges stored in the photodiodes 44 is performed byread-out electronics 52 of the flat panel detector 14, which convert thecharge to digital signals. The digital signals generated by the read-outelectronics 52 may then be provided to an image reconstructor 60 forsubsequent processing and generation of a digital image of the westernblot sample, as indicated at STEP 72, with the digital image displayingspecific proteins in the blot sample so as to allow for further dataanalysis thereof, such as evaluation of protein levels, molecular weightanalysis, and/or another quantitative analysis thereof, for example.

According to another embodiment, the flat panel imaging system 10generally described in FIGS. 1-4 is utilized for fluorescence imaging ofa gel sample. In such an embodiment, the flat panel imaging system 10may be modified as shown in FIGS. 6 and 7 in order to accommodate suchfluorescence imaging. Referring first to FIG. 6, the flat panel imagingsystem 10 is shown therein as including a transillumination light source74 that provides illumination for performing of fluorescence imagingwhen required. In one embodiment—such as would be used in DNA/RNAsequence detection—the transillumination light source 74 is anultraviolet (UV) light source that is incorporated into the lid 18 ofthe housing 12. In another embodiment—such as would be used in proteindetection—the light source 74 is a colored light source (e.g.,red/green/blue (RGB) light source or blue light source) that isincorporated into the lid 18 of the housing 12. The light source 74 isused in conjunction with a light-activated fluorescent stain or reagentto generate a light emission from the gel or blot sample 20. As shown inFIG. 7, a narrow bandwidth filter 76 may be included in the flat panelimaging system 10, with the filter 76 being incorporated into the flatpanel detector 14 to filter the light source emission from light source74 from the light generated by the gel sample responsive to excitationof the fluorescent reagent by the light source. According to anotherembodiment, a combination of a long pass filter and short pass filtercould be used instead of the narrow bandwidth filter 76.

Referring now to FIG. 8, and with continued reference back to FIGS. 1-4and FIGS. 6 and 7, a technique 78 of image generation for fluorescenceimaging of a gel sample is illustrated according to an embodiment of theinvention. The technique begins 78 at STEP 80 with the providing of agel sample 20 (i.e., no membrane) that is prepared in accordance with amanner commonly known in the art. The gel sample 20 is solidified to theextent that it can be provided as a standalone sample placed directlyonto the flat panel detector 14—with the sample typically having ajello-like consistency. The gel sample 20 may be in the form of aDNA/protein sample in agarose or polyacrylamide gel, with gelelectrophoresis being performed to separate proteins/DNA in the sample.

The gel sample 20, and more specifically the macromolecules in the gelsample, are fluorescently labeled to make them visible. A fluorescentreagent is utilized that causes a light to be emitted from the gelsample when the reagent is excited by a light source—such as lightsource 74 (UV or colored light source). The fluorescent reagent may beany of a number of stained nucleic acid gels that can be excited by a UVor colored light source. In one embodiment, where DNA/RNA is analyzedvia excitation with UV light, the reagent may be ethidium bromide(EtBr), iridium bromide, or SYBR® Green, for example. In anotherembodiment, where protein is analyzed via excitation with colored light(or UV), the reagent may be Texas Red or SYPRO® Ruby, for example.Similar to enzyme reactions (as in chemiluminescence), the fluorescentreagents may be optimized for optimal signal-to-noise ratio, as if thedegree of fluorescent labeling is too low, the signal will be weak andif the degree of fluorescent labeling is too high, the signal will alsobe weak due to the inactivation of the detection reagent or quenching ofthe signal caused by a phenomenon known as Forster resonance energytransfer (FRET). Thus, upon applying of the fluorescent reagent, acompleted fluorescence blot sample 20 is provided at STEP 80.

Referring still to FIG. 8, in a next step of technique 78, the gelsample 20 is placed onto the flat panel detector 14 of the imager 10, asindicated at STEP 82. More specifically, the gel sample 20 is placed onthe top protective layer 22 of the flat panel detector 14 such that itis in direct contact therewith. Accordingly, the gel sample 20 ispositioned immediately adjacent to the amorphous silicon photodetectorarray 24 of the flat panel detector 14. Upon placement of the gel sample20 on the flat panel detector 14, the lid 18 of the flat panel imagingsystem 10 is closed at STEP 84 and the light source is activated at STEP86. An output of light from the light source 74 excites thefluorescently labeled sample and causes light to be emitted from the gelsample 20, and this light is acquired/captured by the flat paneldetector 14 at STEP 88. The light emitted from the gel sample 20 that iscaptured by the flat panel detector 14 is converted to electric charge,which is stored in the photodiodes 44 of the photodetector pixels 34 andsubsequently read out by activating the thin film transistors 46associated with the photodiodes 44.

The read-out of the charges stored in the photodiodes 44 is performed byread-out electronics 52 of the flat panel detector 14, which convert thecharge to digital signals. The digital signals generated by the read-outelectronics 52 may then be provided to an image reconstructor 60separate from the flat panel detector 14 for subsequent processing andgeneration of a digital image of the gel sample 20, as indicated at STEP89. For the technique 78 described above, the digital image generated atSTEP 89 will display specific proteins/DNA/RNA in the gel sample so asto allow for further data analysis thereof, such as evaluation ofprotein levels and quantitative analysis thereof.

According to another embodiment, the flat panel imaging system 10generally described in FIGS. 1-4 is utilized for absorbance orcolorimetric (i.e., absorbance) imaging of a gel sample 20. In such anembodiment, the flat panel imaging system 10 may be modified as shown inFIGS. 9 and 10 in order to accommodate such colorimetric imaging.Referring first to FIG. 9, the flat panel imaging system 10 is showntherein as including a transillumination light source 90 to provideillumination for performing of colorimetric imaging. According to oneembodiment, the transillumination light source 90 that is incorporatedinto the lid 18 of the housing 12 is a white light source, and colorfilter arrays 92 may be placed over the photodetector pixels 34 of theflat panel detector 14 to capture the color information, as is shown inFIG. 10. In another embodiment where the light source 90 is a whitelight source, each pixel 34 may be divided into three sub-pixels eachfiltered by a thin-film filter of a specific color—such that each pixelcaptures all three colors (red/green/blue). In such an embodiment, it isrecognized that the displayed image for the gel sample could be a colordisplay that uses the color that has the highest absorbance for the usedstain.

While light source 90 is described above as a light source thatilluminates the full area of the gel sample, it is understood that inanother embodiment the light source 90 could be configured as a sourcethat selectively scans the gel sample using a point, line or patch. Byproviding a light source that scans the gel sample area using a point,line or patch, the contrast in the colorimetric detection can beincreased.

Referring now to FIG. 11, and with continued reference back to FIGS. 1-4and FIGS. 9 and 10, a technique 94 of image generation for colorimetricimaging is illustrated according to an embodiment of the invention. Thetechnique 94 begins at STEP 96 with the providing of a gel sample thatis prepared in accordance with a manner commonly known in the art. Thegel sample 20 is solidified to the extent that it can be provided as astandalone sample placed directly onto the flat panel detector 14—withthe sample typically having a jello-like consistency. As previously setforth in detail above, in providing a sample, proteins are firstseparated using gel electrophoresis and then exposed to a soluble dye tostain the proteins in the gel sample. Examples of soluble dyes that maybe employed to stain the proteins are silver stain and Coomassie stains(e.g., Coomassie® Brilliant Blue dye). Upon a desired staining of theproteins, the gel sample is destained to enable visualization of theproteins, upon completion of which a colorimetric gel sample 20 isprovided at STEP 96.

Referring still to FIG. 11, in a next step of technique 94, the gelsample 20 is placed onto the flat panel detector 14 of the imager 10, asindicated at STEP 98. More specifically, the gel sample 20 is placed onthe top protective layer 22 of the flat panel detector 14 such that itis in direct contact therewith. Accordingly, the gel sample 20 ispositioned immediately adjacent to the amorphous silicon photodetectorarray 24 of the flat panel detector 14. Upon placement of the gel sample20 on the flat panel detector 14, the lid 18 of the flat panel imagingsystem 10 is closed at STEP 100 and the light source 90 is activated atSTEP 102. An output of light (white light or colored light) from thelight source 90 provides for a densitometry (or absorptiometry)measurement to be taken of the gel sample 20 that measures lightabsorption through the gel, with the light absorption measurements beingacquired/captured by the flat panel detector 14 at STEP 104. The lightabsorption through the gel sample 20 that is captured by the flat paneldetector 14 is converted to electric charge, which is stored in thephotodiodes 44 of the photodetector pixels 34 and subsequently read outby activating the thin film transistors 46 associated with thephotodiodes 44.

The read-out of the charges stored in the photodiodes 44 is performed byread-out electronics 52 of the flat panel detector 14, which convert thecharge to digital signals. The digital signals generated by the read-outelectronics 52 may then be provided to an image reconstructor 60separate from the flat panel detector 14 for subsequent processing andgeneration of a digital image of the gel sample 20, as indicated at STEP106. For the technique 94 described above, the digital image generatedat STEP 106 will display specific proteins in the gel sample so as toallow for further data analysis thereof, such as evaluation of proteinlevels and quantitative analysis thereof.

According to additional embodiments of the invention, it is recognizedthat both the fluorescence imaging technique 78 of FIG. 8 and thecolorimetric/absorbance imaging technique 94 of FIG. 11 could beperformed on a blot sample rather than a gel sample. That is,macromolecules (protein/DNA/RNA) could be transferred from the gel ontoa membrane to create a blot sample that is placed in the flat panelimaging system 10 for image acquisition. In such an embodiment—forcolorimetric or chemiluminescence—the member could be wetted such thatthe blot sample becomes semi-transparent or translucent, allowing lightfrom a light source in the imaging system lid to pass through thesample. While such an embodiment can lead to less exciting light passingthrough the sample (as compared to the gel sample embodiment) and lesslight uniformity across the sample/membrane, it is recognized that imageacquisition of a blot sample in chemifluorescence imaging andcolorimetric/absorbance imaging is recognized to be within the scope ofthe invention.

EXAMPLES

The following examples were carried out using a flat panel imagingsystem, such as the flat panel imaging system 10 of FIGS. 1-4. Images ofa western blot were acquired via a flat panel imaging system, and theseimages were compared to images acquired of the same western blot using aCCD-based imaging system and a C-Digit imaging system (LICOR system).

Example 1

In a first example, a sample of 6.25 ng of Actin protein was provided ona western blot—with chemiluminescence detection being used to acquire animage. The Actin protein sample was imaged on each of a flat panelimaging system, a CCD-based imaging system, and a C-Digit imagingsystem. In performing the image acquisitions with the aforementionedsystems, an image of the Actin protein was acquired with the flat panelimaging system using a 12 second acquisition time (i.e., exposure time),an image of the Actin protein was acquired with the CCD system using a12 minute acquisition time, and an image of the Actin protein wasacquired with the C-Digit system using a 12 minute acquisition time. Forthe acquired images, it was found that a crisper image of the Actinprotein was acquired with the flat panel imaging system due to thesensitivity of the flat panel detector and based on the SNR achievablewith the flat panel detector.

With respect to the SNRs present in the imaging systems, the SNR isdetermined for purposes of this example based on a peak signal detectedduring image acquisition of the Actin protein sample and on a standarddeviation of noise across a plurality of pixels in the acquired image(e.g., 100 pixels). The standard deviation of noise, σ_(noise), acrosspixels in the image acquired with the flat panel imaging system is 11,while the σ_(noise) across pixels in the image acquired with the CCDsystem is 35 and the σ_(noise) across pixels in the image acquired withthe C-Digit system is 30. Based on these standard deviations of noiseand the peak signals detected with the respective systems, the SNRsachieved with the respective systems are 305 (+/−10) for the flat panelimaging system, 132 (+/−10) for the CCD system, and 34 (+/−5) for theC-Digit system. Thus, it can be seen that an improved SNR is achievablewith the flat panel imaging system, leading to improved resolution andcrispness in the images of the Actin protein acquired therewith.

Example 2

In a second example, a cell lysate sample of unknown makeup was providedon a western blot, with a sample of Actin protein also provided on thewestern blot to serve as a standard or reference point—withchemiluminescence detection being used to acquire an image. Referringnow to FIG. 12, images acquired of the cell lysate western blot areshown therein, with an image 110 of the western blot acquired via use ofa flat panel imaging system illustrated in comparison to similar imagesacquired via the use of a CCD-based imaging system and a C-Digit imagingsystem, indicated as 112 and 114 respectively. Each of the images 110,112, 114 includes three lanes therein that are generally referred to asa high concentration lane 116, a medium concentration lane 118, and alow concentration lane 120, in which the concentration of the celllysate sample is varied.

For performing an image capture of the cell lysate sample, a line scanacquisition is performed. As can be seen in FIG. 12, a linescan—indicated as 122—is performed on the high concentration lane 116 toprovide a high quality image thereon. As can be seen in image 110, theflat panel imaging system provides increased sensitivity to lightgenerated by the sample along with improved SNR, such that the image 110includes increased focus and contrast of the cell lysate lanes 116, 118,120 shown in the image. With particular respect to high concentrationlane 116, it can be seen that the standard/reference Actin protein isvisible, as indicated at 124, and that another protein in the sample,indicated at 126, is clearly discernible in the lane 116 of image 110.However, in each of the CCD image 112 and the C-Digit image 114, theprotein 126 is not discernible/detectable and. Furthermore, particularlywith respect to the C-Digit image 114, it is seen that “bleeding” ispresent in the image between proteins, based on a large amount ofbackground noise present in the system.

In performing the image acquisitions with the aforementioned systems, animage of the cell lysate sample was acquired with the flat panel imagingsystem using a 8 second acquisition time (i.e., exposure time), an imageof the cell lysate sample was acquired with the CCD system using a 12minute acquisition time, and an image of the cell lysate sample wasacquired with the C-Digit system using a 12 minute acquisition time. Asa standard chemiluminescence reaction on a western blot has a lifetimeof 10-20 minutes, it is thus recognized that only one or two separateexposures and accompanying image acquisitions might be performed withthe CCD system and the C-Digit system, thereby limiting the variationsof exposure time that might be desired by an operator and the number ofseparate images that might be acquired—which may lead to anoversaturated or undersaturated image. Conversely, based on therelatively short exposure time associated with the image acquisition ofthe flat panel imaging system, the system is recognized as havingessentially an infinite dynamic range, as a much larger number ofseparate exposures and accompanying image acquisitions can be performedduring the lifetime of the chemiluminescence reaction, such that anideal exposure time can be identified for optimum image acquisition. Thecollection of numerous images via the fast acquisition rate also allowsfor a decrease in the noise of the acquired images.

Beneficially, embodiments of the invention thus provide a flat panelimaging system 10 having a flat panel detector 14 that functions as amatrix-based light sensor array, with the flat panel detector 14 beingcomposed of an array of pixels each comprising a photodiode-transistorpair that detect/capture light emitted from a gel or blot imagingprocess that utilizes a chemiluminescence, fluorescence or colorimetricdetection technique. Each pixel may be sized so as to provide forreasonable spatial resolution in capturing light from the gel/blotimaging, with pixels down to a size of 50 microns being included in theflat panel detector. The flat panel imaging system provides demandingperformance in terms of sensitivity, dynamic range, exposure time, andquantum efficiency, and collects photons directly and efficiently fromthe gel/blot sample, which eliminates costly high-efficiency imagingoptics used with small cooled CCD image sensors and greatly improves theworkflow associated with traditional film—with image capture times ofless than 10 seconds (e.g., 6 seconds) being achievable. The increase insensitivity also allows for dramatically decreasing the amount of sampleneeded, thus reducing anti-body, reagents required, and laboratoryanimals, saving costs and time, while the fast acquisition speed enablesthe use of software to obtain virtually infinite dynamic range, reducingtime and effort for each experiment. Of still further benefit, the flatpanel imaging system also can provide a quasi-stationary image withreasonable signal-to-noise ratio, which is superior to a scan method.The flat panel imaging system also offers compactness for portability.

Therefore, according to one embodiment, a method for generating adigital image in fluorescence detection gel imaging includes providing agel sample having a gel and a sample of macromolecules therein andplacing the gel sample on a flat panel detector of a flat panel imagingsystem, the flat panel detector comprising an array of photodiodes andtransistors that collect light generated from the gel sample. The methodalso includes illuminating the gel sample using a light sourceintegrated into the flat panel imaging system and collecting lightemitted by the gel sample responsive to an excitation of the gel sampleby light provided by the light source, with the light emitted by the gelsample being collected by the array of photodiodes of the flat paneldetector and converted to electric charges to generate light data. Themethod further includes processing the light data to generate a digitalimage of the gel sample, the processing and image generation beingperformed by an image reconstructor separate from or incorporated intothe flat panel imaging system.

According to another embodiment, a method of generating a digital imagein fluorescence detection gel imaging includes providing a gel samplelabeled with a fluorescent reagent, the gel sample having macromoleculeslabeled by the fluorescent reagent. The method also includes positioningthe gel sample within a flat panel imaging system to provide forcapturing of a digital image of the gel sample, the positioning of thegel sample within the flat panel imaging system further includingplacing the gel sample on a flat panel matrix-based light sensorcomprising an array of photodiodes and transistors and closing a lid ofthe flat panel imaging system to create a closed environment forcapturing of the digital image. The method further includes illuminatingthe gel sample using a light source integrated into the lid of the flatpanel imaging system so as to excite the fluorescent reagent causing thegel sample to generate fluorescent light and detecting the fluorescentlight emitted by the gel sample using the flat panel matrix-based lightsensor, with the fluorescent light being collected by the array ofphotodiodes, converted to electric charges, and subsequently convertedto digital signals. The method still further includes providing thedigital signals to an image reconstructor to process the digital signalsand generate a digital image of the gel sample.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

What is claimed is:
 1. A method for generating a digital image incolorimetric detection gel imaging, the method comprising: providing agel sample comprising a gel and a sample of macromolecules therein;placing the gel sample on a flat panel detector of a flat panel imagingsystem, the flat panel detector comprising an array of photodiodes andtransistors that collect light generated from the gel sample;illuminating the gel sample using a light source integrated into theflat panel imaging system; collecting light transmitted through the gelsample that is not absorbed by the gel sample, the light transmittedthrough the gel sample being collected by the array of photodiodes ofthe flat panel detector and converted to electric charges to generatelight data; processing the light data to generate a digital image of thegel sample, the processing and image generation being performed by animage reconstructor separate from or incorporated into the flat panelimaging system; and filtering the light transmitted through the gelsample by way of a color filter, so to provide for collection of colorinformation by the array of photodiodes; wherein the color filtercomprises a removable filter that forms part of a protective layerpositioned at a top surface of the flat panel detector.
 2. The method ofclaim 1 wherein placing the gel sample on the flat panel detectorcomprises applying the gel sample directly onto a protective layer thatforms a top surface of the flat panel detector.
 3. The method of claim 2wherein collecting light emitted by the gel sample comprises collectinglight emitted by the gel sample that is transmitted through theprotective layer to the array of photodiodes, the protective layerhaving a thickness of 25-75 urn so as to prevent light spreading andmaintain a desired spatial resolution of the light emitted by the gelsample so as not to degrade a modulation transfer function (MTF) of theflat panel detector.
 4. The method of claim 1 wherein the flat paneldetector comprises an array of pixels each including a photodiode andassociated transistor, with the array of pixels being arranged in aplurality of rows and columns.
 5. The method of claim 1 wherein the gelsample comprises a gel sample having 1D separation of proteins achievedvia gel electrophoresis of the gel sample.
 6. The method of claim 5wherein illuminating the gel sample comprises transilluminating the gelsample using a white light source integrated into the flat panel imagingsystem.
 7. The method of claim 1 wherein the color filter comprises athin-film filter of a specific color positioned over each of a pluralityof sub-pixels included in each pixel of the flat panel detector, thethin-film filter of a specific color comprising one of a red-green-bluefilter.
 8. The method of claim 1 wherein illuminating the gel samplecomprises selectively scanning the gel sample using a point, line orpatch light source, so as to provide for increased contrast in thecolorimetric detection.
 9. The method of claim 1 wherein collectinglight transmitted through the gel sample comprises collecting lighttransmitted through the gel sample for a duration of 10 seconds or lessto acquire and generate light data sufficient to generate a digitalimage of the gel sample.
 10. The method of claim 1 wherein the flatpanel matrix-based light sensor comprises one of amorphous silicon,poly-silicon, an organic photodiode, or a crystal silicon technology.11. A method of generating a digital image in colorimetric detection gelimaging, the method comprising: providing a gel sample including a geland a sample of macromolecules therein, the macromolecules being stainedwith a soluble dye; positioning the gel sample within a flat panelimaging system to provide for capturing of a digital image of the gelsample, the positioning of the gel sample within the flat panel imagingsystem including: placing the gel sample on a flat panel matrix-basedlight sensor comprising an array of photodiodes and transistors; andclosing a lid of the flat panel imaging system to create a closedenvironment for capturing of the digital image; illuminating the gelsample using a light source integrated into the lid of the flat panelimaging system; measuring a light absorption through the gel sampleusing the flat panel matrix-based light sensor, with the lightabsorption through the gel sample being measured by the array ofphotodiodes, converted to electric charges, and subsequently convertedto digital signals; and providing the digital signals to an imagereconstructor to process the digital signals and generate a digitalimage of the gel sample.
 12. The method of claim 11 wherein placing thegel sample on the flat panel matrix-based light sensor comprisesapplying the gel sample directly onto a protective layer that forms atop surface of the flat panel matrix-based light sensor, the protectivelayer constructed to prevent light spreading and enable a desiredspatial resolution of the light emitted by the gel sample.
 13. Themethod of claim 11 wherein illuminating the gel sample comprisestransilluminating the gel sample using a white light source integratedinto the flat panel imaging system; and wherein the generated digitalimage of the gel sample provides a visualization of proteins in the gelsample.
 14. The method of claim 11 further comprising filtering thelight absorption through the gel sample by way of a color filter, so toprovide for collection of color information by the array of photodiodes.15. The method of claim 14, wherein the color filter comprises athin-film filter of a specific color positioned over each of a pluralityof sub-pixels included in each pixel of the flat panel detector, thethin-film filter of a specific color comprising one of a red-green-bluefilter.
 16. The method of claim 14, wherein the color filter comprises aremovable filter that forms part of a protective layer positioned at atop surface of the flat panel detector.
 17. The method of claim 11wherein illuminating the gel sample comprises selectively scanning thegel sample using a point, line or patch light source, so as to providefor increased contrast in the colorimetric detection.
 18. The method ofclaim 11 wherein measuring the light absorption through the gel samplecomprises measuring the light absorption through the gel sample for aduration of 10 seconds or less sufficient to generate a digital image ofthe gel sample.
 19. The method of claim 11 wherein the flat panelmatrix-based light sensor comprises an array of pixels that each includea respective photodiode and transistor, with the array of pixels havinga pitch of 50 μm or greater so as to provide a reasonable spatialresolution for detecting the light emitted by the sample.
 20. The methodof claim 11, wherein placing the gel sample on the flat panelmatrix-based light sensor comprises applying the gel sample directlyonto a protective layer that forms a top surface of the flat panelmatrix-based light sensor.
 21. The method of claim 11, wherein the gelsample comprises a gel sample having 1D separation of proteins achievedvia gel electrophoresis of the gel sample.
 22. A method for generating adigital image in colorimetric detection gel imaging, the methodcomprising: providing a gel sample comprising a gel and a sample ofmacromolecules therein; placing the gel sample on a flat panel detectorof a flat panel imaging system, the flat panel detector comprising anarray of photodiodes and transistors that collect light generated fromthe gel sample; illuminating the gel sample using a light sourceintegrated into the flat panel imaging system; collecting lighttransmitted through the gel sample that is not absorbed by the gelsample, the light transmitted through the gel sample being collected bythe array of photodiodes of the flat panel detector and converted toelectric charges to generate light data; processing the light data togenerate a digital image of the gel sample, the processing and imagegeneration being performed by an image reconstructor separate from orincorporated into the flat panel imaging system; wherein placing the gelsample on the flat panel detector comprises applying the gel sampledirectly onto a protective layer that forms a top surface of the flatpanel detector; and wherein the protective layer includes an anglediscriminating film to increase contrast and decrease crosstalk ofphotons received by the array of pixels.
 23. A method for generating adigital image in colorimetric detection gel imaging, the methodcomprising: providing a gel sample comprising a gel and a sample ofmacromolecules therein; placing the gel sample on a flat panel detectorof a flat panel imaging system, the flat panel detector comprising anarray of photodiodes and transistors that collect light generated fromthe gel sample; illuminating the gel sample using a light sourceintegrated into the flat panel imaging system; collecting lighttransmitted through the gel sample that is not absorbed by the gelsample, the light transmitted through the gel sample being collected bythe array of photodiodes of the flat panel detector and converted toelectric charges to generate light data; processing the light data togenerate a digital image of the gel sample, the processing and imagegeneration being performed by an image reconstructor separate from orincorporated into the flat panel imaging system; wherein collectinglight transmitted through the gel sample and processing the light datafurther comprises: discharging the electric charges stored in the arrayof photodiodes by activating the transistors; and performing a read-outof the discharged electric charges via read-out electronics in the flatpanel detector to convert the electric charges to digital signals,thereby generating the light data.